Potential of chimeric antigen receptor (CAR)‐redirected immune cells in breast cancer therapies: Recent advances

Abstract Despite substantial developments in conventional treatments such as surgery, chemotherapy, radiotherapy, endocrine therapy, and molecular‐targeted therapy, breast cancer remains the leading cause of cancer mortality in women. Currently, chimeric antigen receptor (CAR)–redirected immune cell therapy has emerged as an innovative immunotherapeutic approach to ameliorate survival rates of breast cancer patients by eliciting cytotoxic activity against cognate tumour‐associated antigens expressing tumour cells. As a crucial component of adaptive immunity, T cells and NK cells, as the central innate immune cells, are two types of pivotal candidates for CAR engineering in treating solid malignancies. However, the biological distinctions between NK cells‐ and T cells lead to differences in cancer immunotherapy outcomes. Likewise, optimal breast cancer removal via CAR‐redirected immune cells requires detecting safe target antigens, improving CAR structure for ideal immune cell functions, promoting CAR‐redirected immune cells filtration to the tumour microenvironment (TME), and increasing the ability of these engineered cells to persist and retain within the immunosuppressive TME. This review provides a concise overview of breast cancer pathogenesis and its hostile TME. We focus on the CAR‐T and CAR‐NK cells and discuss their significant differences. Finally, we deliver a summary based on recent advancements in the therapeutic capability of CAR‐T and CAR‐NK cells in treating breast cancer.


| INTRODUC TI ON
Breast cancer is a heterogeneous disorder with the highest prevalence among malignancies, and it is still one of the top causes of cancer death in women around the world. Breast cancer is divided into molecular subsets with distinctive biology and clinical characteristics. 1,2 Every year, almost 2 million new breast cancer cases are identified worldwide, and over half a million individuals die from the disease due to recurrence or metastasis. [3][4][5][6] Early detection and significant advances in standard conventional modalities (e.g., chemotherapy, radiotherapy, surgery, and hormone therapy) have improved cure rates and quality of life in women with localized breast cancer.
At the same time, some subsets with distant metastases remain the primary concern for treatment. [7][8][9][10] Consequently, using innovative therapeutic modalities in the treatment of breast cancer is urgently required to open up new avenues to apply novel therapeutic targets to decrease the disease's recurrence and death rates.
Cancer immunotherapy, which exploits immune cells' natural anticancer capacities, has emerged as one of the most promising options for potentiating the process of cancer cell elimination. 11 Adoptive cell-based antitumor therapy has become a landmark event and a rapidly developing modality for cancer-targeted therapy in recent decades. 12,13 Dendritic cells (DCs), T cells, and alloreactive natural killer (NK) cells are some of the immune effector cells used in cancer cellular therapy. 14 As a crucial component of adaptive immunity, T cells and NK cells, as the primary innate immune cells, have received tremendous interest in cancer therapy, mainly contributing to cancer elimination and immune surveillance. 15,16 However, genetic and epigenetic modifications in the tumour microenvironment associated with tumour cell evasion from immune responses cause antitumor immune responses to be delayed, changed, or even resistant, permitting tumour development. [17][18][19][20][21] Meanwhile, breast cancer may evade immune surveillance and increase malignant persistence through various mechanisms. They include the recruitment of regulatory T cells and myeloid-derived suppressor cells (MDSCs) to the TME, changes in the expression of NK cell activating receptors that affect their interaction with other cells, neutralization of T cell effectors, expressing immune checkpoints, and alteration in the capacity of myeloid dendritic cells and plasmacytoid DCs. 18,22,23 Scientists have devised techniques to redirect immune effector cells and thus boost anticancer properties, concomitantly inhibiting immune escape from circumventing these mechanisms. As a result, recombinant constructs known as chimeric antigen receptors (CARs) have been used to genetically engineer T and NK immune effector cells to improve adoptive cellular therapy and tumoricidal activities. 24,25 CARs are synthetic surface receptors that have been broadly used to redirect T and NK cells and can recognize a specific target antigen on the surface of cancer cells. CARs activate redirected effector cells and eventually tumour cell lysis upon detection. The basic CAR construct comprises a single-chain variable fragment (scFv; ectodomain) that serves as an extracellular antigen-recognition domain and is linked to a diversity of intracellular signalling domains (endodomain). 13,26,27 CAR proteins can recognize a broad range of MHC-independent tumour antigens, allowing them to attack more tumour cells. 28-3 0 Meanwhile, adoptive transfer of redirected T and NK cells expressing CAR has demonstrated empowering outcomes in treating a variety of haematological malignancies. In contrast, a comparable impact on solid tumours has not been observed. 31,32 However, detecting a relevant target antigen and using complementary genetic strategies to protect these redirected cells from immunosuppressive signals delivered within the tumour microenvironment (TME) could pave the way to use this technique in solid tumours, particularly breast cancer. 9,23 The current review first discusses the pathogenesis and function of effector cells in the breast cancer microenvironment, followed by a discussion of recent findings in CAR T cell therapy and CAR NK cell therapy in breast cancer, with a particular emphasis on last decade reports.

NIKOO et al.
Breast cancer family history is an essential factor of disease risk.
Around 20-25% of patients have a positive family history, and only 5-10% of all breast cancers are related to gene mutations inherited from a parent. [37][38][39] Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) tumour-suppressor genes have been identified as two significant susceptibility genes in breast cancer, with mutations in the BRCA1 and BRCA2 genes involved at least 30% of hereditary breast cancer cases. 37,40 In addition to BRCA1 and BRCA2, germline mutations in five additional susceptibility genes, including tumour protein P53 (TP53), phosphatase and tensin homologue (PTEN), checkpoint kinase 2 (CHEK2), ataxia telangiectasia mutated (ATM), and partner and localizer of BRCA2 (PALB2), have been recognized as cancerrelated genes in breast cancer patients. [41][42][43][44] Germline mutations in the TP53 gene cause Li-Fraumeni syndrome, with a high chance of developing early-onset breast cancer. Furthermore, mutations in the PTEN genes, which cause Cowden syndrome, and serine/ threonine kinase 11 (STK11), which identifies as a causative gene in Peutz-Jeghers syndrome, have been linked to an increased risk of breast cancer. Investigations have shown that pathogenic mutations in BRCA1/BRCA2 increase the risk of BC by 10-to 20 fold. Besides, mutations in TP53 also give a high chance of BC, so a mutation in the TP53 was found in 65-80% of basal or TNBC breast cancers. 45,46 According to a meta-analysis of BRCA1 and BRCA2 carrier families, the lifetime risk of breast cancer varies from 65% to 81% for BRCA1 and 45% to 85% for BRCA2. Another genetic variation associated with intermediate dangers of breast cancer and a 20%-40% lifetime chance of getting breast cancer includes the CHEK2, ATM, and PALB2 genes involved in the DNA repair process. 33 Analysing mutations with correct and reliable genetic testing in the significant genes (BRCA1 and BRCA2) and less commonly mutated genes (such as PTEN) and subsequent genetic counselling in high-risk women can be beneficial in the early detection and/or prevention of breast cancer development ( Table 1).
There are several kinds of breast cancer, each defined by unique units of the breast and specific cells that are affected. Most breast cancers result from an oncogenic transformation of the epithelial compartment of breast tissue (carcinoma), which comprises cells that line functional units of lobules and terminal mammary ducts.
Sarcomas, such as phyllode tumours and angiosarcomas, are a small subset of breast cancer (1% of primary breast cancer) that arise from alteration of the connective tissue compartment of breast tissue, which consists of myofibroblasts and blood vessel cells. 47,48 Breast carcinoma, the most common type of breast cancer, progresses through three major stages: non-invasive (or in situ), invasive, and metastatic. Non-invasive or pre-invasive treatment is limited to the epithelium component of pre-existing normal ducts. Because this stage has a high potential for progression to invasive carcinoma, early detection and prompt and proper therapy are paramount in preventing progression to the invasive form. Invasive carcinoma has broken through and infiltrated the epithelial components of the breast lobules and ducts, migrating into the surrounding breast connective tissue. Although it is possible to eradicate invasive carcinoma from its primary origin of development, invasive breast cancer has the potential to spread to other organs of the body, such as lymph nodes and/or distant organs such as the lung, liver, bone, and brain, and progress to metastatic breast cancer. The risk of breast carcinoma metastasizing is not easily detected, and around 30% of women with primary-stage breast carcinoma will progress to the metastatic stage of the disease. 33,47,49 Recent advances in gene expression profiling techniques have significantly influenced our understanding of breast cancer biology. 50,51 Gene expression studies have highlighted many different molecular breast cancer subtypes related to breast cancer biology and demonstrate considerable variations in their incidence, risk factors, prognosis, and therapeutic responses. 52 The distinction between molecular/intrinsic subtypes of breast cancer is based on a diversity of inherent genes, including hormone-related genes, human epidermal growth factor receptor 2 (HER2)-related genes, proliferation-related genes, and the basal cluster of genes. 44,53,54 Breast tumours are classified into five molecular/intrinsic subtypes based on gene expression patterns of this cluster of genes (e.g., Luminal-A, Luminal-B, HER2-enriched, basal-like, and normal breast-like). 55

| B RE A S T C AN CER TRE ATMENT
There are currently no benefit treatment options for TNBC patients and approved targeted therapies are ineffective in these patients.
Typical breast cancer has similar characteristics to luminal A disorder, but its prognosis is somewhat worse than that of the luminal A subtype. 33  (IFNγ) production. [81][82][83][84] Cancer-associated fibroblasts (CAFs) are another immunosuppressive cell found in the TME that generate TGFβ and vascular endothelial growth factor (VEGF) and suppress T cell activity.
Moreover, studies have demonstrated that removing CAFs from the TME in breast cancer decreases the recruitment of TAMs, MDSCs, and T regulatory cells and reduces tumour angiogenesis and lymphangiogenesis. [85][86][87] Given that T cells play a pivotal role in the adap-

| IMMUNE CELL SOURCE S FOR C AR-BA S ED TARG E TED THER APY IN B RE A S T C AN CER
In recent years, genetically modifying immune cells to express CARs has represented a novel adoptive cell therapy strategy in treating various progressive cancers. The genetic modification of functional T and NK cells depends on efficient and permanent gene transfer. 15,24,32 Immune cells can be acquired from numerous cell sources using leukapheresis, discussed further below.

| THE IMP ORTAN CE OF ANTI G EN S ELEC TI ON
The first step in developing effective CAR therapies for breast cancer is to select appropriate target antigens. Choosing the right CAR

| OVERVIE W OF RECENT S TUD IE S BA S ED ON C AR-T CELL S IN B RE A S T C AN CER
The amount and quality of research in breast cancer immunotherapy directed at CAR T cells has dramatically risen in recent years. 164 Substantial efforts are being made to improve the efficacy of CAR T therapy against solid tumours, including the identification of appropriate target antigens, evolvement of the next-generation CAR T cells with improved capabilities, increasing the efficiency of T cell responses to moderate T cell dysfunction in the suppressive TME, and developing new strategies to overcome restrictions in tumour T cell trafficking. [165][166][167] As previously mentioned, CAR T cells identify several forms of target antigens, whose proteins are among the most antigenic targets changed or overexpressed on the surface of malignant cells. Carbohydrates and glycolipids are two more CAR T-cell targets often modified in tumour cells 168,169 (Table 2).

Folate receptor-alpha (FR) is a glycosylphosphatidylinositol
(GPI)-linked surface protein highly overexpressed in non-mucinous epithelial malignancies such as ovarian, breast, and lung tumours.
FR has been overexpressed in specific malignancies, such as ERnegative, stage IV metastatic TNBC, at roughly 86%. However, its expression in other breast cancer subtypes is only 30%, making FR an appealing target for breast cancer immunotherapy. 170 Table 3).

| CLINI C AL TRIAL S FOR C AR-T CELL THER APY IN B RE A S T C AN CER
Numerous researches have been carried out at the preclinical in vitro and in vivo levels of treatment with CAR-T cells in breast cancer, which has progressed to first-in-human studies, as shown in Table 4. A phase I trial was initiated to assess the safety and optimal dose of intraventricularly administered autologous HER2-targeted

| CON CLUS I ON AND FUTURE DIREC TION
There is an unmet therapeutic necessity to develop effective thera- Several obstacles related to CAR-redirected immune cell treatment in breast cancer must be considered to make this strategy safer and more effective. One of the critical issues is identifying optimal antigenic targets in breast cancer with a deregulated expression on both primary tumour cells and cells resident in the TME (e.g., MDSCs, TAMs, CAFs, Tregs) overcoming tumour escape. [204][205][206]   Writing -review and editing (equal). Morteza Akbari: Supervision (lead).

ACK N OWLED G EM ENTS
We appreciate Dr. Amin Daei Sorkhabi (Tabriz University Of Medical sciences, Tabriz, Iran) and Dr. Aila Sarkesh (Tabriz University of Medical sciences, Tabriz, Iran) for their partial contribution in the first draft of the manuscript.

CO N FLI C T O F I NTE R E S T
There is no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.